Electrotransfer of naked DNA in the skeletal muscles of animal models of muscular dystrophies.

The electrotransfer of naked DNA has recently been adapted to the transduction of skeletal muscle fibers. We investigated the short- and long-term efficacy of this methodology in wild-type animals and in mouse models of congenital muscular dystrophy (dy/dy, dy(2J)/dy(2J)), or Duchenne muscular dystrophy (mdx/mdx). Using a reporter construct, the short-term efficacy of fiber transduction reached 40% and was similar in wild-type, dy/dy and dy(2J)/dy(2J) animals, indicating that ongoing muscle fibrosis was not a major obstacle to the electrotransfer-mediated gene transfer. Although the complete rejection of transduced fibers was observed within 3 weeks in the absence of immunosuppression, the persistency was prolonged over 10 weeks when transient or continuous immunosuppressive regimens were used. Using therapeutic plasmids, we demonstrated that electrotransfer also allowed the transduction of large constructs encoding the laminin alpha2 chain in dy/dy mouse, or a chimeric dystrophin-EGFP protein in mdx/mdx mouse. The correct sarcolemmal localization of these structural proteins demonstrated the functional relevance of their expression in vivo, with a diffusion domain estimated to be 300 to 500 microm. However, degeneration-regeneration events hampered the long-term stability of transduced fibers. Given its efficacy for naked DNA transfer in these models of muscular dystrophies, and despite some limitations, gene electrotransfer methodology should be further explored as a potential avenue for treatment of muscular dystrophies.

Neuromuscular function impairment is not caused by motor neurone loss in FALS mice: an electromyographic study.

Dominant mutations of human Cu/Zn superoxide dismutase (SOD1) are found in about 20% of patients with familial amyotrophic lateral sclerosis (FALS). A transgenic mouse model of FALS (FALSG93A mice) has been generated by overexpression of a mutated form of SOD1. Using electromyography we first show that FALSG93A mice suffer from motoneurone dysfunction similar to that observed in ALS patients and fulfill Lambert's criteria for ALS. We also showed that FALSG93A mice demonstrate a massive loss of functional motor units starting at 47 days of age. Impairment of motor neurone function preceeds by 6 weeks the onset of apparent clinical signs (shaking, tremor) and the beginning of motor neurone loss. Neuromuscular deficits in FALS mice do not result from motoneuronal cell death but rather from loss of axonal integrity.

Electromyographical and motor performance studies in the pmn mouse model of neurodegenerative disease.

The mouse autosomal recessive mutation progressive motor neuronopathy (pmn) results in early onset motor neuron disease with rapidly progressive hindlimb paralysis, severe muscular wasting, and death at around 6 weeks of age. This mutant provides opportunities for testing novel therapeutic strategies, including the administration of trophic factors, to prevent the degeneration of diseased neurons. The construction of a strain expressing the pmn and the Extra-toe (Xt) phenotypes allows the detection, and therefore the treatment, of affected progeny before the onset of the clinical weakness. Electromyography is the most appropriate technique for a longitudinal study in which a given individual is examined repeatedly. We present the results of an electrophysiological and behavioral exploration of the pmn disease and show that electromyography is a powerful tool for following the course of the disease and evaluating potential therapies relevant to motor neuron diseases.

Myasthenia gravis: comparative autoantibody assays using human muscle, TE671, and glucocorticoid-treated TE671 cells as sources of antigen.

The use of acetylcholine receptors (AChR) from the readily available TE671 cell line as a practical alternative to human muscle for monitoring the anti-AChR antibody assay in sera of Myasthenia gravis patients has been recently proposed. Most of the TE671 culture protocols include the use of glucocorticoids. Glucocorticoids were shown to upregulate the acetylcholine receptor expression in TE671 cells. To confirm the advantage of using AChR from TE671 cells (AChRTE) and to validate the use of AChR from glucocorticoid-treated cells (AChRGT) in AChR antibody measurement, the three different antigens (muscle AChR (AChRMU), AChRTE, and AChRGT) were compared for radioimmunoprecipitation assay. We found that, despite a slight underestimation of the antibody titers using AChRTE and AChRGT compared to AChRMU, and considering the rare cases of AChRMU antibody titer category permutations, the correlations between the values were satisfactory.

alpha-Bungarotoxin sensitization in experimental autoimmune myasthenia gravis.

A mouse model of MG, termed experimental autoimmune myasthenia gravis (EAMG), can be obtained after immunization with Torpedo acetylcholine receptor (AChR). Although many studies have detailed the consequence of AChR antibodies binding at the neuromuscular junction and the difficulty in obtaining obvious clinical signs, less attention has been focused on the possibility of amplifying the muscular block in order to discriminate between immunized and healthy animals. In the present studies we observe that a single inoculation of alpha-bungarotoxin (alpha-bgt) can amplify the neuromuscular block revealed by repetitive nerve stimulation, and induce in EAMG mice a stable muscular weakness state lasting for at least 169 hours instead of 95 hours in normal mice. This model could provide an excellent tool for evaluating drugs active on neuromuscular transmission.